原代神經細胞培養方法NeuronCellCulture

1. Preparation of coverslips

1.1- Mass culture

Our standard mass cultures are plated on astrocytes.  Those, in turn, are plated on glass coverslips pre-coated with poly-D-lysine and laminin.

Materials:

. #1 coverslips

. coverslip racks in a water-tight container (we made ours)

. poly-D-lysine (PDL) stock solution (1mg/ml in dd water)

. laminin stock solution (20 mg/ml in Hank’s BSS)

. 35 mm plastic culture dishes

. culture hood equipped with UV lamp

. sterile dd water

Procedure:

. Place the coverslips in the racks and leave them in the culture hood under UV light for 2 hrs.

. Coat the coverslips with 12.5 mg/ml PDL (5ml PDL in 400ml sterile dd water) for 2hrs. in the culture hood.

. Wash the coverslips with sterile dd water five times, in the culture hood.

. Place the coverslips in the sterile 35mm dishes

. Add 0.4ml laminin on top of the coverslips. Wait for 45’, then aspirate the excess solution

1.2- Agarose-collagen microislands

 This protocol is based on protocols by Segal and Furshpan. Although the following “macro-island” approach has allowed for greater neuronal survival, while still providing a high probability of connection between DRG and dorsal horn (DH) autapses or  DH-DH connections can only be obtained with high probability in the conventional microislands.

Materials:

. #1 coverslips coated with PDL as above

. type II agarose

. Vitrogen 100 collagen, ~3 mg/ml

. 35 mm plastic culture dishes

. culture hood equipped with UV lamp

. sterile dd water

. atomizer

Procedure:

. Place coverslips in 35 mm dishes

. Melt agarose in dd water at 0.2%, and place a drop on the top surface of each coverslip. The height of each drop is diminished as much as possible by removing excess solution with a pipette before the agarose gels.

. Allow coated coverslips to air dry overnight in the culture hood at room temperature to form a thin film. For adequate drying, the dishes must be uncovered.

. Spray the collagen onto the coverslips with the atomizer.  We use a glass perfume bottle which can be bought at Macy’s inNew York. In our experience that makes bigger droplets than the Fisher chromatography atomizer, and is much less expensive (and looks better too).  The atomizer is held parallel to the bottom of the dishes, about 25 cm above and away from them.  It is then pumped forcefully a few times.

. The collagen islands can be examined with an inverted microscope.  Their size should be between 300 and 1000 mm in order to maximize the probability of connection between any two neurons.

. ADD A DROP OF COLLAGEN to the edge of the coverslip.  That will serve as support for a “feeder culture” that helps the survival of the insular neurons

. The collagen is also allowed to dry overnight, under ultraviolet light.

1.3- “Macro”-islands

We find that few dorsal horn neurons seem to survive on the “traditional”  atomizer-generated microislands, apparently regardless of neuron density.  Although we still do not know the reason for that, we developed a variation of the microisland method which generates bigger (est. 1-3 mm) islands.  With this method, the probability of finding an usable island in a giver coverslip is very much increased.  Although, with such big islands, interconnected DH neurons are about as easy to find as in a mass culture, DRG neurons, which seem to grow extremely long axons, are usually connected to most of the DH neurons present.

Materials:

same as in 2.2, except:

.instead of the atomizer, a fine painting brush (#3 or smaller)

Procedures:

. All identical to 2.2, except for the collagen spraying.

. Dip the paint brush in the collagen solution, then shake the excess solution off the brush.

. Gently tap the brush on the edge of each dish, rotating the dish a few degrees after each tap.

. Control the quality of the islands by looking at the dishes through an inverted microscope.  Some practice is required to optimize the islands.

2. Dissection, plating, and maintenance of cells

2.1- Media

In our system, Neurobasal + B-27 seems to improve the survival of the neurons, as well as increase neurite extension/branching. However, it does not favor astrocyte survival.  Overall, we tend to use it for the co-cultures in “macro-islands” and for the mass cultures in general, but not for the microislands on astrocytes.

A. IMDM for Astrocytes

Prepare stocks:

. For Glucose/Glutamine/Pen-Strep solution, mix

60 g of glucose in 200 ml dd water

100 ml Glutamine 200 mM

100 ml Penniciline-Streptomycine

. Filter through a 0.22 mm filter, separate in 20 aliquots of 20 ml each and freeze

. For FVM, mix

15 mg 6,7-dimethyl-5,6,7,8-tetrahydropterine hydrochloride

75 mg glutathione

1.5 g ascorbic acid

into 300 ml dd water